Method for generating transcriptionally active DNA fragments

ABSTRACT

A method for producing transcriptionally active DNA molecules, comprising (PCR) amplification of said DNA molecule in the presence of a first DNA fragment (F1), second DNA fragment (F2), first primer (P1), a second primer (P2), a third primer (P3), and a fourth primer (P4) wherein: F1 comprises a promoter sequence; F2 comprises a terminator sequence; P1 is complementary to the 5′ end of F1; P2 is complementary to the 5′ end of F2; P3 comprises a first region complementary to the 3′ end of F1 and a second region complementary to the 5′ end of said DNA molecule; P4 comprises a first region complementary to the 3′ end of F2 and a second region complementary to the 3′ end of said DNA molecule.

RELATED APPLICATIONS

[0001] The present application is a continuation of U.S. patentapplication Ser. No. 09/535,262, filed Mar. 23, 2000, and claimspriority to U.S. Provisional Application Serial No. 60/125,953, filedMar. 24, 1999, both of which are hereby expressly incorporated byreference in their entireties.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a method for generatingtranscriptionally active DNA fragments. More specifically, the methodrelates to synthesis of a DNA fragment by polymerase chain reaction(PCR) using nested primers, promoter sequences and terminator sequences.

[0004] 2. Description of the Related Art

[0005] In addition to the tremendous progress made in the past few yearsin sequencing the human genome, efforts have also been made to sequenceother organisms that are of biomedical importance. For example, completegenomic sequences have been obtained for Borrelia burgdorferi (cause ofLyme disease), Chlamydia, Heliobacter pylori and Mycobacteriumtuberculosis. The fast-growing sequence information provides immenseopportunities to reveal the basic biology of related organisms at thegene/molecular level and to develop novel therapeutics or vaccinesagainst various pathogens.

[0006] However, this vast sequence information also mandates a much moreefficient and streamlined way to screen and identify genes of interestfrom tens of thousands of candidate genes. The conventional approach togene screening and identification involves generation of a cDNA library,subcloning the DNA inserts into plasmid vectors (expression vectors),purifying plasmid DNA from bacteria for each individual cDNA clone andtransfecting animal cells or tissues for functional analysis of theencoded gene product. This method, even in conjunction with the use ofpolymerase chain reaction (PCR) to generate cDNA fragments to allowdirectional and in-frame cloning, is still time consuming, costly anddifficult to automate.

[0007] The present invention provides a simple, rapid method for thegeneration of transcriptionally active DNA fragments.

SUMMARY OF THE INVENTION

[0008] One embodiment of the present invention is a method forgenerating a transcriptionally active DNA molecule, comprisingpolymerase chain reaction (PCR) amplification of said DNA molecule inthe presence of a first DNA fragment (F1), second DNA fragment (F2),first primer (P1), a second primer (P2), a third primer (P3) and afourth primer (P4) wherein: F1 comprises a promoter sequence; F2comprises a terminator sequence; P1 is complementary to the 5′ end ofF1; P2 is complementary to the 3′ end of F2; P3 comprises a first regioncomplementary to the 3′ end of F1 and a second region complementary tothe 5′ end of said DNA molecule; P4 comprises a first regioncomplementary to the 5′ end of F2 and a second region complementary tothe 3′ end of said DNA molecule, whereby a transcriptionally active DNAmolecule is produced by said PCR amplification. Preferably, F1 is thecytomegalovirus IE promoter. In one aspect of this preferred embodiment,the transcriptionally active DNA molecule encodes a therapeutic gene.The method may further comprise the step of adding a PNA tail to the5′-end of P1 and P2 prior to the PCR amplification. Preferably, athymidine base immediately precedes the region of complementaritybetween the third primer P3 and the first DNA fragment F1. In anotheraspect of this preferred embodiment, a thymidine base immediatelyprecedes the region of complementarity between the fourth primer P3 andthe second DNA fragment F2. The method may also further comprise thestep of adding a PNA clamp to said transcriptionally active DNA moleculeafter said PCR amplification. Preferably, the method further comprisesthe step of adding a PNA molecule via a linker (PNA clamp tail) toprimers P1 and P2 prior to the PCR amplification.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 is a schematic diagram of the gene amplification method ofthe present invention. Oligonucleotide primers P1 and P2 arecomplementary to the 5′ end of a promoter sequence (F1) and the 3′ endof a terminator sequence (F2), respectively. P3 and P4 areoligonucleotide primers which contain regions complementary to oppositeends of Gene X. In addition, P3 and P4 contain regions which arecomplementary to the 3′ region of F1 and the 5′ region of F2. An excessamount of primers P1 and P2 are combined with Gene X, or with a mixtureof DNA containing Gene X, and with F1, F2, P3 and P4, or with a mixtureof DNA containing F1, F2 and Gene X which has been PCR amplified usingP3 and P4, and subjected to PCR to produce a transcriptionally activelinear DNA fragment containing F1, F2 and gene X.

[0010]FIG. 2 is a schematic diagram showing DNA fragment F1 whichcontains a thymidine base immediately preceding the region ofcomplementarity with the PCR intermediate (primer P3).

[0011]FIG. 3 is a schematic diagram showing the use of peptide nucleicacid (PNA) sequences and PNA clamps to protect the ends of linearexpression DNA fragments. Primers P1 and P2 contain a PNA tail which isresistant to proteolytic and exonuclease degradation. The resultinglinear expression DNA fragment contains a PNA tail at each of the5′-ends. The 3′ ends are protected by addition of a PNA “clamp” which isalso resistant to proteolytic and exonuclease degradation.

[0012]FIG. 4 is a schematic diagram showing the use of a PNA “clamp”tail to protect the 5′ ends of the linear expression DNA fragment,followed by formation of a clamp during PCR to protect the 3′ ends.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention provides a simple, efficient method forgenerating transcriptionally active DNA fragments which can be readilytransfected into animal cells or tissues by conventional nucleic acidtransfection techniques, without the need for subcloning into expressionvectors and purification of plasmid DNA from bacteria. Thetranscriptionally active DNA fragments are synthesized by polymerasechain reaction (PCR) amplification of any gene of interest using nestedoligonucleotide primers and two DNA fragments, one of which comprises anactive transcription promoter sequence and one of which comprises abasic transcription terminator element. A first primer is complementaryto the DNA fragment comprising the promoter; a second primer iscomplementary to the DNA fragment comprising the terminator; a thirdprimer is complementary to both the promoter sequence and one end of thegene of interest; and a fourth primer is complementary to both theterminator sequence and the other end of the gene of interest. Thesepromoters, genes and terminators are linked in an expression cassette asshown in FIG. 1. In addition, the ends of linear DNA containing theexpression cassette can be protected from exonuclease digestion duringand after transfection by incorporating peptide nucleic acid (PNA)sequences and PNA clamps as shown in FIGS. 3 and 4.

[0014] As used herein, the term “promoter” is a DNA sequence whichextends upstream from the transcription initiation site and is involvedin binding of RNA polymerase. The promoter may contain several short(<10 base pair) sequence elements that bind transcription factors,generally dispersed over >200 base pairs. A promoter that contains onlyelements recognized by general and upstream factors is usuallytranscribed in any cell type. Such promoters may be responsible forexpression of cellular genes that are constitutively expressed(sometimes called housekeeping genes). There are also tissue-specificpromoters limited to particular cell types, such as the humanmetallothionein (MT) promoter which is upregulated by heavy metal ionsand glucocorticoids.

[0015] As used herein, the term “terminator” is a DNA sequencerepresented at the end of the transcript that causes RNA polymerase toterminate transcription. This occurs at a discrete site downstream ofthe mature 3′ end which is generated by cleavage and polyadenylation.

[0016] The present method can be used to quickly generatenuclease-resistant and transcriptionally active linear DNA moleculeswhich express any desired gene with any known sequence. The linear DNAcan then be delivered into animal cells or tissues for functionalanalysis, vaccination and other pharmaceutical applications. This methodalso avoids problems associated with bacterial growth such as toxicityand stability. This method can also be completely automated for use inhigh-throughput screening methods.

[0017] Referring now to FIG. 1, oligonucleotide primers P1 and P2 arecomplementary to the 5′ region of DNA fragments F1 and F2, respectively.Fragment F1 comprises a transcription promoter (darkened region) andfragment F2 comprises a transcription terminator (darkened region).Primers P3 and P4 are complementary to the 3′ ends of F1 and F2,respectively, and to different ends of the DNA fragment containing GeneX. FIG. 1 shows the putative intermediates and the final product whichare formed in the present PCR-based method when P1, P2, P3, P4 , F1 andF2 are added to a DNA template comprising Gene X. It should be notedthat amount of primers P1 and P2 present in the reaction mixture arepreferably at least about 100 fold greater than the amounts of P3, P4,F1 and F2, to minimized the amounts of intermediate products generatedduring the reaction. To generate the first intermediate, primer P3 andP4 are used which amplify from the promoter and terminator sequences ofF1 and F2 across Gene X, resulting in an intermediate having thepromoter and terminator sequences on the ends and Gene X in betweenthese sequences.

[0018] The second intermediate is formed by hybridization of the firstintermediate with fragments F1 and F2 via their complementary promoterand terminator sequences (darkened regions), followed by PCRamplification from primers P3 and P4, resulting in a DNA segmentcomprising the entire F1, F2 and Gene X.

[0019] In the last step of the reaction, PCR amplification of the secondintermediate using primers P1 and P2 results in amplified amounts of thecomplete transcriptionally active DNA fragment.

[0020] The method of the invention may be performed by adding all of thecomponents in a single reaction mixture. Alternatively, two separate PCRreactions can be performed. In the first reaction, the template gene, P3and P4 are used first. The product of this reaction is then used as atemplate for a second PCR reaction involving fragments F1 and F2, plusprimers P1 and P2.

[0021] The generation of final products using the nested PCR methoddescribe above is dependent on the sequences of the F1 or F2 fragmentsat the junction between the region overlapping P3 and P4, respectively.This is due, at least in part, to the addition of an extra adenosinebase (A) at the 3′ end of the PCR fragment by Taq DNA polymerase whichis commonly used in PCR protocols. This could produce a mismatch betweenthe PCR intermediate generated by P3/P4 and fragments F1 and F2.

[0022] Thus, in a preferred embodiment, the overlap between the PCRintermediate (P3 primer) and F1, and P4/F2, is designed such that athymidine base (T) immediately precedes the overlap region in fragmentsF1 and F2 (FIG. 2, only F1 shown). In this case, even with the additionof an “A” base to the 3′ end to the intermediate during PCRamplification, the overlap between F1/F2 and the respective PCRintermediates is still perfectly maintained.

[0023] Peptide nucleic acids (PNA) are nucleic acid analogs in which theentire deoxyribose-phosphate backbone has been exchanged with achemically completely different, but structurally homologous, polyamide(peptide) backbone containing 2-aminoethyl glycine units. Unlike DNA,which is highly negatively charged, the PNA backbone is neutral.Therefore, there is much less repulsive energy between complementarystrands in a PNA-DNA hybrid than the comparable DNA-DNA hybrid, andconsequently they are much more stable.

[0024] In addition, molecules called PNA “clamps” have been synthesizedwhich have two identical PNA sequences joined by a flexible hairpinlinker containing three 8-amino-3,6-dioxaoctanoic acid units. When a PNAclamp is mixed with a complementary homopurine or homopyrimidine DNAtarget sequence, a PNA-DNA-PNA triplex hybrid can form which isextremely stable (Bentin et al., Biochemistry 35:8863-8869, 1996; Egholmet al., Nucleic Acids Res. 23:217-222, 1995; Griffith et al., J. Am.Chem. Soc. 117:831-832, 1995). The sequence-specific and high affinityduplex and triplex binding of PNA have been extensively described(Nielsen et al., Science 254:1497-1500, 1991; Egholm et al., J. Am.Chem. Soc. 114:9677-9678, 1992; Egholm et al., Nature 365:566-568, 1993;Almarsson et al., Proc. Natl. Acad. Sci. U. S. A. 90:9542-9546, 1993;Demidov et al., Proc. Natl. Acad. Sci. U. S A. 92:2637-2641, 1995). Theyhave also been shown to be resistant to nuclease and protease digestion(Demidov et al., Biochem. Pharm. 48:1010-1013, 1994).

[0025] A representative PNA clamp is shown in FIGS. 3 and 4. This PNAclamp, or a PNA clamp having any desired DNA sequence, is synthesized asdescribed by Egholm et al. (supra.) or is available from PerSeptiveBiosystems (Framingham, Mass.). The representative PNA clamp shown inFIGS. 2 and 3 has the sequence 5′-TCTCTCTC-O-O-O-TJTJTJTJ (SEQ ID NO:1), where J (pseudoisocytosine) is an analog of C, and the “O” residuesare 8-amino-3,6-dioxaoctanoic acid linkers which separate the tworegions of the PNA.

[0026] The use of PNA tails, PNA clamps and PNA “clamp tails” to protectthe ends of the transcriptionally active PCR fragments which aredescribed above is illustrated in FIG. 2. The ends of the PCR fragmentsgenerated as shown in FIG. 1 are prone to digestion by exonucleasesafter transfection into a cell or tissue. Two strategies for inhibitingthis degradation are shown in FIGS. 3 and 4. In FIG. 1, a PNA tail and ashort DNA sequence (5′-GATC-O-TCTCTCTC-3′; SEQ ID NO: 2) is added to the5′ end of primers P1 and P2 which bind to the target sequence5′-GAGAGAGA-3′ (SEQ ID NO: 3). This generates a PNA binding site at the3′ end of the opposite strand during PCR. Primers containing PNA tailscan be synthesized using methods well known in the art or are availablefrom PerSeptive Biosystems. These PNA tails do not hybridize to a targetsequence, but protect the 5′-ends from exonuclease digestion. In orderto protect the 5′ ends of the PCR fragment, a PNA molecule is added tothe 3′-PNA tail-protected PCR fragment. This PNA contains a DNA sequencewhich is complementary to the 3′-end of the PCR fragment and bindsthereto as shown in FIG. 3.

[0027] An alternate PNA protection approach is shown in FIG. 4. In thismethod, a PNA clamp is linked to primers P1 and P2 via one or more8-amino-3,6-dioxaoctanoic acid linkers to form a PNA “clamp” tail bywell known methods. After PCR amplification, the “clamp tail” binds tothe 3′ ends of the PCR fragment, and is linked to the 5′ ends via theone or more 8-amino-3,6-dioxaoctanoic acid linkers to protect both the3′ and 5′ ends of the PCR fragment.

[0028] Although the use of the CMV IE promoter and an artificialmammalian transcriptional terminator elements are exemplified herein,the use of any eukaryotic promoter and terminator is within the scope ofthe present invention. Suitable promoters for use in the presentinvention include, for example, SV40, rous sarcoma virus (RSV),retroviral long terminal repeats (LTR), muscle creatine kinase promoter,actin promoter, elongation factor promoter, synthetic promoters,tissue-specific promoters, and the like. Suitable terminator sequencesinclude SV40 transcription terminator, bovine growth hormone (BGH)terminator, synthetic terminators, and the like. These promoter andterminator sequences can be obtained by restriction enzyme digestion ofcommercially available plasmids and cDNA molecules, or can besynthesized using an automated DNA synthesizer using methods well knownin the art.

[0029] Any desired gene may be amplified and coupled to active promoterand terminator sequences using the method of the present invention. In apreferred embodiment, the gene encodes a gene product which is absent orpresent at reduced levels in an organism. Nonlimiting examples of thesegene products are the cystic fibrosis transmembrane regulator (CFTR),insulin, dystrophin, interleukin-2, interleukin-12, erythropoietin,gamma interferon, and granulocyte macrophage colony stimulating factor(GM-CSF).

[0030] Although any transfection method well known in the art may beused to transfect the transcriptionally active PCR fragments of theinvention into cells or tissues including calcium phosphateprecipitation, electroporation and DEAE-dextran, cationic lipid-mediatedtransfection is preferred. Gene delivery systems are described byFelgner et al. (Hum. Gene Ther. 8:511-512, 1997) and include cationiclipid-based delivery systems (lipoplex), polycation-based deliverysystems (polyplex) and a combination thereof (lipopolyplex). Cationiclipids are described in U.S. Pat. Nos. 4,897,355 and 5,459,127, theentire contents of which are hereby incorporated by reference.

EXAMPLE 1 Production of Transcriptionally Active PCR Fragments

[0031] The following components are combined in a 100 μl reactionvolume: DNA fragment F1 (4 ng) which comprises a region of hightranscriptional potency (−240 to +60) from the human cytomegalovirus(CMV) immediate early gene (IE) promoter/enhancer, DNA fragment F2 (4ng), a 55 base pair oligonucleotide encoding an artificial mammaliantranscription terminator element(5′-CACAAAAAACCAACACACAGATCTCTAGAGCTCTGATCTTTTATTAGCCA GAAGT-3′; SEQ IDNO: 4), 400 ng primer P1 (5′-TCTCTCTACGTATTAGTCATCG-3′; SEQ ID NO: 5),400 ng primer P2 (5′-TCACAAAAAACCAACACACAG-3′; SEQ ID NO: 6), 4 ngprimer P3 and 4 ng primer P4. The P1 and P2 primer sequences correspondto the 5′ end of fragment F1 and the 5′ end of fragment F2, respectively(FIG. 1). Primers P3 and P4 are designed based on the gene sequence ofinterest. The 3′ potion of these primers (about 10-20 base pairs) isdetermined by the actual sequence of the gene to be amplified, whereasthe 5′ region of P3 comprises the following sequence:5′-CTCCGCGGATCCAGA-3′ (SEQ ID NO: 7) to overlap with the 3′ region ofF1, and P4 comprises the sequence 5′-TTATTAGCCAGAAGT-3′ (SEQ ID NO: 8)to overlap with the 3′ region of F2.

[0032] PCR is performed as follows: denaturation for 30 seconds at 94°C., annealing for 45 seconds at 55° C. and extension for three minutesat 72° C. for 25-30 cycles. The size of the final produce is verified by1% agarose gel electrophoresis. The amplified PCR fragment is cleanedand purified using a commercial PCR cleaning kit (e.g., Qiagen), and canbe used for in vitro or in vivo transfection of cells or tissues.

EXAMPLE 2

[0033] The green fluorescent protein (GFP, 700 bp) was used as a targetgene. The F1 fragment was a 600 bp fragment of the CMV immediate earlygene promoter (from −550 to +50). A 50 bp oligonucleotide containing amodified rabbit beta-globin gene transcription terminator was used asprimer 2. Two different versions of these primers were designed so thatthe overlap between F1 and the intermediate GFP PCR fragment was eitherpreceded with or without a thymidine base. After the PCR reaction wascarried out according to the conditions described in Example 1, theproducts were analyzed by electrophoresis. When a thymidine basepreceded the overlap region in F1, a clean major PCR fragment of about1.4 kb was produced, representing the GFP coding region flanked by CMVpromoter (F1) and the transcription terminator, whereas if a base otherthan thymidine preceded the overlapping region, no product wasgenerated. The fragment generated by nested PCR was then transfectedinto Cos-7 cells and the expression of GFP was monitored by fluorescencemicroscopy. The results showed that the intensity and frequency ofGFP-expressing cells was almost the same between cells transfected withthe PCR fragment and a supercoiled plasmid DNA in which the same CMVpromoter was driving expression of the GFP gene. Thus, the presentmethod produces DNA fragments which are transcriptionally active.

[0034] While particular embodiments of the invention have been describedin detail, it will be apparent to those skilled in the art that theseembodiments are exemplary rather than limiting, and the true scope ofthe invention is that defined in the following claims.

1 8 1 16 DNA Artificial Sequence Synthetic oligonucleotide primer 1tctctctctn tntntn 16 2 12 DNA Artificial Sequence Syntheticoligonucleotide primer 2 gatctctctc tc 12 3 8 DNA Artificial SequenceSynthetic oligonucleotide primer 3 gagagaga 8 4 55 DNA ArtificialSequence Synthetic oligonucleotide primer 4 cacaaaaaac caacacacagatctctagag ctctgatctt ttattagcca gaagt 55 5 22 DNA Artificial SequenceSynthetic oligonucleotide primer 5 tctctctacg tattagtcat cg 22 6 21 DNAArtificial Sequence Synthetic oligonucleotide primer 6 tcacaaaaaaccaacacaca g 21 7 15 DNA Artificial Sequence Synthetic oligonucleotideprimer 7 ctccgcggat ccaga 15 8 15 DNA Artificial Sequence Syntheticoligonucleotide primer 8 ttattagcca gaagt 15

What is claimed is:
 1. A method for amplifying atranscriptionally-active polynucleotide, comprising: PCR-amplifying afirst fragment of DNA with a first primer pair, wherein the first primerpair, upon such amplification, adds to first and second ends of thefirst fragment predetermined first and second regions ofcomplementarity, to form a second DNA fragment having said first regionof complementarity at a first end and a second region of complementarityat a second end of said second DNA fragment; providing apromoter-containing sequence and a terminator-containing sequence, saidpromoter-containing sequence further including a region complementary tosaid first region of complementarity, and said terminator-containingsequence further including a region complementary to said second regionof complementarity, wherein both said promoter-containing sequence andsaid terminator-containing sequence include an internal nucleotidecapable of forming an A-T base pair immediately adjacent to said regionof complementarity; joining said promoter-containing sequence to saidfirst end of said second DNA fragment and said terminator-containingsequence to said second end of said second DNA fragment to form saidthird DNA fragment; and PCR-amplifying said third DNA fragment.
 2. Themethod of claim 1, wherein said joining comprises joining in thepresence of polymerase said promoter-containing sequence to said firstend of said second DNA fragment and said terminator-containing sequenceto said second end of said second DNA fragment to form said third DNAfragment.
 3. The method of claim 1, wherein said promoter-containingsequence and said terminator-containing sequence further comprise anon-DNA, binding moiety capable of interacting with said second DNAfragment.
 4. The method of claim 3, wherein said non-DNA, binding moietycomprises a PNA molecule.